Analysis of Environmental and Pathogenic Bacteria Attached to Aerosol Particles Size-Separated with a Metal Mesh Device.

Metal mesh devices (MMDs) are novel materials that enable the precise separation of particles by size. Structurally, MMDs consist of a periodic arrangement of square apertures of characteristic shapes and sizes on a thin nickel membrane. The present study describes the separation of aerosol particles using palm-top-size collection devices equipped with three types of MMDs differing in pore size. Aerosols were collected at a farm located in the suburbs of Nairobi, Kenya; aerosol particles were isolated, and pathogenic bacteria were identified in this microflora by next-generation sequencing analysis. The composition of the microflora in aerosol particles was found to depend on particle size. Gene fragments were obtained from the collected aerosols by PCR using primers specific for the genus Mycobacterium. This analysis showed that Mycobacterium obuense, a non-tuberculous species of mycobacteria that causes lung diseases, was present in these aerosols. These findings showed that application of this MMD analytical protocol to aerosol particles can facilitate the investigation of airborne pathogenic bacteria.

Preparation and characterization of glycopolymers with biphenyl spacers via Suzuki coupling reaction.

Poly(vinylbiphenyl)s bearing glycoside ligands at the side chains were prepared using the Suzuku coupling reaction. Effects of glycoside reactant concentration, halide species, glycoside species, and catalyst species on the incorporation of glycoside ligand into the polymer were investigated. The obtained glycopolymers exhibited specific binding to proteins corresponding to the glycoside ligands. In addition, the biphenyl spacers formed by the Suzuki coupling reaction in the glycopolymer were fluorescent, whereas the polymer precursor was not.

Bio-inert Properties of TEG Modified Dendrimer Interface.

The bioinert interfaces that prevent adhesion of proteins and cells are important for biomaterial applications. In order to design a bioinert interface, the immobilization of an appropriate functional group and the control of molecular density is required. Dendrimer was modified with triethylene glycol (TEG) to display a dense brush structure. TEG with different density and terminal groups were immobilized with a dendrimer template and thiol terminated molecules. The inhibitory effect on protein and bacteria binding was investigated. The physical property of the interface was measured by QCM-admittance to clarify the factor of the bioinert property.

Formation of glyco-functionalized interfaces for protein binding using polyphenolic glycoside.

In this study, a polyphenolic glycoside (α-glucosyl rutin) was used to form glyco-functionalized interfaces for protein binding. α-Glucosyl rutin was coated onto precious metals, metal oxides, and synthetic polymers, including polyethylene and polytetrafluoroethylene with poor surface modifiability. The glyco-functionalized interfaces bound strongly and specifically to concanavalin A and Bauhinia purpurea lectin, which have different carbohydrate specificities. Competitive adsorption tests demonstrated that the binding sites for the abovementioned lectins were glucosyl and rhamnosyl residues, respectively. The glyco-functionalized interfaces maintained the protein binding ability after being stored in aqueous solution for 1 day and in air for 160 days. Once the glyco-functionalized interfaces were formed on gold, silicon dioxide, polystyrene, and polytetrafluoroethylene using α-glucosyl rutin, all the glyco-functionalized interfaces bound to concanavalin A rather than peanut agglutinin.

Biopolymer monolith for protein purification.

Porous glycopolymers, "glycomonoliths", were prepared by radical polymerization based on polymerization-induced phase separation with an acrylamide derivative of α-mannose, acrylamide and cross-linker in order to investigate protein adsorption and separation. The porous structure was induced by a porogenic alcohol. The pore diameter and surface area were controlled by the type of alcohol. The protein adsorption was measured in both batch and continuous flow systems. The glycomonoliths showed specific interaction with the sugar recognition protein of concanavalin A, and non-specific interaction to other proteins was negligible. The amount of protein adsorption to the materials was determined by the sugar density and the composition of the glycomonoliths. Fundamental knowledge regarding the glycomonoliths for protein separation was obtained.

Effect of interfacial serum proteins on the cell membrane disruption induced by amorphous silica nanoparticles in erythrocytes, lymphocytes, malignant melanocytes, and macrophages.

It is very important to examine carefully the potential adverse effects of engineered nanoparticles (NPs) on human health and environments. In the present study, we have investigated the impact of interfacial serum proteins on the cell membrane disruption induced by silica NPs of primary diameter of 55-68 nm in four types of cells (erythrocytes, Jurkat, B16F10, and J774.1). The silica-induced membranolysis was repressed by addition of 1-2% serum into culture media, where the adhesion amount of the FBS-coated silica NPs onto a cell surface seemed comparable with that of the bare silica NPs. The nonspecific attraction between the bare silica and J774.1 cell membrane surfaces was masked by pretreatment of the silica surface with serum albumin, whereas the serum proteins-coated silica surface exhibited the attractive interactions with the cell membrane due to specific binding between some of adsorbed proteins thereon and the membrane receptors. The difference in silica-cell interaction between the nonspecific and specific attractions would explain the reason why interfacial serum proteins reduced the membranolysis without prevention of silica NPs adhering to cell surfaces.

Amplification of Sensor Signals from Metal Mesh Device with Fine Periodic Structure.

Two types of metal mesh devices with hole diameters of 1.7 and 0.3 µm were prepared by an electroforming method. The metal mesh devices with hole diameters of 1.7 and 0.3 µm transmitted electromagnetic waves with frequencies of approximately 100 and 285 THz, respectively. These spectral frequencies shifted depending on the adsorption amount of protein. The slope in the linear relationship between the adsorption amount and spectral shift (i.e. sensitivity) of the metal mesh device with a hole diameter of 0.3 µm was seven times as great as that of the device with a hole diameter of 1.7 µm. These results agreed with the theoretical concept of the sensitivity for the metal mesh device sensor being proportional to the square of the transmittance frequency. As biosensors, the structurally refined metal mesh devices amplified the output signals.

Verification of the Universal Versatility of a Quantitative Protein Measurement Technique Using a Metal Mesh Device.

When proteins are attached to microstructures such as a metal mesh device, changes in their optical properties occur. These changes have been characterized based on actual measurements in the infrared region of the spectrum. We have previously theoretically and experimentally demonstrated the optical changes associated with streptavidin. Here, we investigate three types of proteins: avidin, BSA, and lysozyme. The three proteins were adsorbed onto three types of metal mesh devices having different resonant frequencies, and the corresponding spectra were measured in the infrared region. The change in the frequency of the dip point in the spectrum was extracted to quantitatively determine the quantity of protein; these results were correlated with the quantitative measurements obtained by electrophoresis. By examining three types of different proteins, it was verified that a variety of proteins can be measured based on the optical characteristics of metal mesh devices.

Regulating Detectable Optical Domain in Sensing Technology Using Metal Mesh Devices and Detection of Submicron-size Particles.

A metal mesh device has a structure in which through-holes of the same shape are periodically placed on a thin metal film, and the selection of such a structure makes it possible to sense objects of various sizes. In this study, we showed the structure of the metal mesh device and the relationship between the detectable optical domain and the size of the objects to be measured. In addition, from measurement of changes in electromagnetic wave transmission characteristics of the metal mesh device due to specific adsorption of particles with a mean diameter of 100 nm with surface modification with Streptavidin to a metal mesh device fixed with biotin, we showed that even large particles can be sensed. Based on these examinations, we showed that, by using a metal mesh device with detectable optical domain corresponding to the size of objects, even objects that are larger than protein can be sensed.

Actual consumption amount of personal care products reflecting Japanese cosmetic habits.

Safety assessments of cosmetics are carried out by identifying possible harmful effects of substances in cosmetic products and assessing the exposure to products containing these substances. The present study provided data on the amounts of cosmetic products consumed in Japan to enhance and complement the existing data from Europe and the United States, i.e., the West. The outcomes of this study increase the accuracy of exposure assessments and enable more sophisticated risk assessment as a part of the safety assessment of cosmetic products. Actual amounts of products applied were calculated by determining the difference in the weight of products before and after use by approximately 300 subjects. The results of the study of skincare products revealed that in comparison with the West, large amounts of lotions and emulsions were applied, whereas lower amounts of cream and essence were applied in Japan. In the study of sunscreen products, actual measured values during outdoor leisure use were obtained, and these were lower than the values from the West. The study of the use of facial mask packs yielded data on typical Japanese sheet-type impregnated masks and revealed that high amounts were applied. Furthermore, data were obtained on cleansing foams, makeup removers and makeup products. The data from the present study enhance and complement existing information and will facilitate more sophisticated risk assessments. The present results should be extremely useful in safety assessments of newly developed cosmetic products and to regulatory authorities in Japan and around the world.

Quantitative Measurement of Protein Using Metal Mesh Device.

Biosensing of protein adsorption with metal mesh device (MMD) was investigated by computational calculations and experiments. Electromagnetic field computation was carried out with a single unit cell of MMD. Equivalent circuit model of MMD on the single unit cell was assumed, and the biosensing with MMD was analyzed in detail by computational calculation and experimental measurements. The dip frequency of MMD was shifted by adsorption of protein on MMD. The shift of dip frequency of MMD was proportional to the amount of protein adsorption. The sensitivity of MMD biosensing was dependent on the microstructure of MMD, and proportional to the square of the dip frequency. The refinement of MMD structure can improve the sensitivity of protein detection.

Macroporous Gel with a Permeable Reaction Platform for Catalytic Flow Synthesis.

We mimic a living system wherein target molecules permeate through capillary and cells for chemical transformation. A monolithic porous gel (MPG) was easily prepared by copolymerization of gel matrix, tertiary amine, and cross-linking monomer in one-step synthesis. Interconnected capillaries existed in the MPG, enabling flow application with high permeability. Because the capillaries were constituted of polymer gel, Pd(0)-loaded MPG provided another permeable pathway to substrates in a gel network, contributing to its much high turnover number after 30 days of use, compared with that of Pd(0)-loaded inorganic supports. Interestingly, the gel network size of the MPG influenced the catalytic frequency. Diffusivities of the substrates and product in the gel networks increased with increasing network sizes in relation to catalytic activities. The MPG strategy provides a universal reactor design in conjunction with a practical process and precisely controlled reaction platform.

Glycopolymer monoliths for affinity bioseparation of proteins in a continuous-flow system: glycomonoliths.

In this study, macroporous materials, called glycomonoliths, were produced from saccharide-containing monomers, and used for affinity bioseparation of proteins in a continuous-flow system. The porous structure formation of the glycomonoliths involved polymerization-induced phase separation of the polyacrylamide unit. The pore size could be controlled between several hundred nanometers and several micrometers by changing the alcohol used as the porogenic solvent during the preparation of the monolith. The glycomonolith pores allowed for the permeation of solutions through the monoliths, which meant that they could be used in a continuous-flow system. The adsorption capacities of the glycomonoliths for the saccharide-binding protein (concanavalin A) were larger than that of a glycopolymer-grafted material because of the higher saccharide densities in the monoliths than the grafted material. When concanavalin A was eluted from the glycomonolith, the concentration of concanavalin A in the effluent was up to 11 times higher than that in the feed solution. The adsorption of concanavalin A to the glycomonolith was specific, even in the presence of other proteins.

Inhibition of Bacterial Adhesion on Hydroxyapatite Model Teeth by Surface Modification with PEGMA-Phosmer Copolymers.

Modification of the interface properties on hydroxyapatite and tooth enamel surfaces was investigated to fabricate bacterial resistance in situ. A series of copolymers containing pendants of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and ethylene glycol methacrylate phosphate (Phosmer) were polymerized by conventional free radical polymerization and changing the feed ratio of monomers. The copolymers were immobilized on hydroxyapatite and tooth enamel via the affinity of phosphate groups to hydroxyapatite to form the stable and durable polymer brushes on the surfaces. The amounts of polymer immobilized depended on the phosphate group ratio in the copolymers. Surface modification altered the interfacial properties of hydroxyapatite and inhibited bacterial adhesion. Copolymers containing 40-60% PEGMA segments showed a significant inhibitory effect on bacterial adhesion of S. epidermidis both in the presence and absence of plaque model biomacromolecules.

Evaluation of combinations of in vitro sensitization test descriptors for the artificial neural network-based risk assessment model of skin sensitization.

The skin sensitization potential of chemicals has been determined with the use of the murine local lymph node assay (LLNA). However, in recent years public concern about animal welfare has led to a requirement for non-animal risk assessment systems for the prediction of skin sensitization potential, to replace LLNA. Selection of an appropriate in vitro test or in silico model descriptors is critical to obtain good predictive performance. Here, we investigated the utility of artificial neural network (ANN) prediction models using various combinations of descriptors from several in vitro sensitization tests. The dataset, collected from published data and from experiments carried out in collaboration with the Japan Cosmetic Industry Association (JCIA), consisted of values from the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA), SH test and antioxidant response element (ARE) assay for chemicals whose LLNA thresholds have been reported. After confirming the relationship between individual in vitro test descriptors and the LLNA threshold (e.g. EC3 value), we used the subsets of chemicals for which the requisite test values were available to evaluate the predictive performance of ANN models using combinations of h-CLAT/DPRA (N = 139 chemicals), the DPRA/ARE assay (N = 69), the SH test/ARE assay (N = 73), the h-CLAT/DPRA/ARE assay (N = 69) and the h-CLAT/SH test/ARE assay (N = 73). The h-CLAT/DPRA, h-CLAT/DPRA/ARE assay and h-CLAT/SH test/ARE assay combinations showed a better predictive performance than the DPRA/ARE assay and the SH test/ARE assay. Our data indicates that the descriptors evaluated in this study were all useful for predicting human skin sensitization potential, although combinations containing h-CLAT (reflecting dendritic cell-activating ability) were most effective for ANN-based prediction.

Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals.

To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h-CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS-based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h-CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water-soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance.

Label-free detection of antigen protein using a metal mesh device surface-modified by an antibody.

A biosensor for protein detection was developed using antibody-immobilized metal mesh devices. Antihemoglobin antibodies were covalently immobilized on a metal mesh device. Extraordinary transmission with a dipped structure was observed for a metal mesh device immobilized with antihemoglobin antibodies as well as for the original metal mesh device. Hemoglobin in the mixture solution containing albumin at a hundred-fold concentration was detectable using antihemoglobin-immobilized MMDs. The detectability using the antihemoglobin-immobilized metal mesh device was similar to that of a commercially-available kit for the qualitative determination of hemoglobin.

Metal mesh device sensor immobilized with a trimethoxysilane-containing glycopolymer for label-free detection of proteins and bacteria.

Biosensors for the detection of proteins and bacteria have been developed using glycopolymer-immobilized metal mesh devices. The trimethoxysilane-containing glycopolymer was immobilized onto a metal mesh device using the silane coupling reaction. The surface shape and transmittance properties of the original metal mesh device were maintained following the immobilization of the glycopolymer. The mannose-binding protein (concanavalin A) could be detected at concentrations in the range of 10(-9) to 10(-6) mol L(-1) using the glycopolymer-immobilized metal mesh device sensor, whereas another protein (bovine serum albumin) was not detected. A detection limit of 1 ng mm(-2) was achieved for the amount of adsorbed concanavalin A. The glycopolymer-immobilized metal mesh device sensor could also detect bacteria as well as protein. The mannose-binding strain of Escherichia coli was specifically detected by the glycopolymer-immobilized metal mesh device sensor. The glycopolymer-immobilized metal mesh device could therefore be used as a label-free biosensor showing high levels of selectivity and sensitivity toward proteins and bacteria.

Signal amplified two-dimensional photonic crystal biosensor immobilized with glyco-nanoparticles.

A two-dimensional, glycopolymer-immobilized, photonic crystal (PhC) biosensor was developed for the detection of proteins. Glycopolymers with different conformations, homopolymers and sugar-incorporating nanoparticles were immobilized on the PhC using intermediate succinimide-containing polymers and proteins. The surface modification was analyzed in detail, and the sugar-protein interaction was detected by monitoring changes in the reflection intensity that was expressed by the two-dimensional PhC. The surface modifications were performed successfully, and specific interactions were detected between the glycopolymers and the proteins. Stronger bonds were present between the glycopolymers and the target proteins than between the glycopolymers and the monovalent sugar, because of a clustering effect. The sugar-incorporating nanoparticles showed a larger binding capacity compared with the homopolymers, and low protein concentrations (with a detection limit of 6.0 ng mL-1) were detected using the sugar-incorporating nanoparticle-immobilized PhC. The detection limit of the developed biosensor was lower than that of surface plasmon resonance sensor (1.43 µg mL-1). The results of this study indicated the potential of the developed biosensor for the detection of a variety of biomolecules.